System and method for performing in vivo imaging and oxymetry and FT microscopy by pulsed radiofrequency electron paramagnetic resonance

Abstract

A system for performing pulsed RF FT EPR spectroscopy and imaging includes an ultra-fast excitation subsystem and an ultra-fast data acquisition subsystem. Additionally, method for measuring and imaging in vivo oxygen and free radicals or for performing RF FT EPR spectroscopy utilizes short RF excitations pulses and ultra-fast sampling, digitizing, and summing steps.

What is claimed is: 
     
       1. A fast response pulsed radiofrequency (RF) electron paramagnetic resonance (EPR) system, with the system utilizing a system clock signal, comprising: a pulse generating sequential, non-overlapping transmit, diplexer, and receive gating pulses   an ultra-fast excitation pulse forming subsystem including: an RF signal generator for providing an RF signal having a frequency of between about 200 MHz and about 400 MHz;   a beam splitter, coupled to the output of the RF signal generator for splitting said RF signal into a reference RF signal and an excitation signal RF signal;   a phase shifter, coupled to said beam splitter to receive said transmitted RF signal, for controllably either passing or phase-shifting said RF excitation signal by 180°;   a gating circuit, coupled to said phase shifter and including a gate coupled to receive a transmit gating pulse from said pulse generator having a duration of about 10 to 90 nanoseconds, for transmitting a received RF excitation signal when said transmit gating pulse is asserted, to form an excitation pulse having a duration of about 10 to about 90 nanoseconds with rise times of less than about 2 nanoseconds;     an ultra-fast data acquisition system including: a gated preamplifier, having a signal input port and having a control input coupled to receive a receive gating pulse, said gated preamplifier amplifying RF radiation received at said signal input port only when said receive gating pulse is received and said gated preamplifier being isolated from RF radiation received at said signal input port when said receive gating pulse is not received, with said gated preamplifier for amplifying EPR response RF radiation received at said signal input port to form an EPR response signal;   demodulating means, coupled to receive said reference RF signal and said EPR response signal, for demodulating said EPR response signal to form an EPR parameter signal;   an ultra-fast, sampling and summing unit, coupled to said demodulating means, for averaging a series of EPR parameter signals to increase signal to noise ratio, said sampling and summing unit including a high-speed sampler to digitize each received EPR parameter signal and a summing means, coupled to receive each digitized EPR parameter signal, for generating a running sum of said digitized EPR parameter signals;     a resonator for inducing paramagnetic resonance in a sample when an excitation pulse is received, for detecting EPR response RF radiation emitted from the sample due to paramagnetic resonance, and for outputting EPR response RF radiation;   a diplexer, coupled to said pulse generator to receive said excitation pulse, coupled to said resonator to receive the EPR response RF radiation, coupled to the signal input port of said gated preamplifier, and having a control input for receiving a diplexer gating pulse of a preset duration, said diplexer for coupling said ultra-fast pulse forming subsystem to said resonator when said diplexer gating pulse is received, for isolating said pulse forming system from said ultra-fast data acquisition system when said diplexer gating pulse is not received, and for providing said EPR response RF radiation from the resonator to the input signal port of said gate preamplifier subsequent to receiving said diplexer gating pulse.   
     
     
       2. The system of claim 1 wherein said resonator is characterized by a Q parameter, where the bandwidth of the resonator response is inversely-proportional to the magnitude of Q and the resonator ring-down time is proportional to Q, said system further comprising: Q-switching means, coupled to said resonator and said timing controller to receive a Q-switching pulse, for increasing resonator Q and decreasing ring-down time for said resonator when a Q-switching pulse is asserted;   and wherein said pulse generator generates a Q-switching pulse of about 20 nanoseconds immediately after said transmit pulse is received at said resonator.   
     
     
       3. The system of claim 1 further comprising: a DC magnet field for generating a constant magnetic field to induce magnetization in said sample;   a gradient magnet for forming a gradient in said constant magnetic field.   
     
     
       4. A method for measuring EPR parameters utilized to perform in vivo measurement or imaging of oxygen tension in a living sample, with a gated RF amplifier for amplifying response radiation generated by the sample, said method comprising the steps of: providing a paramagnetic contrast agent which interacts with in vivo oxygen in the living sample to increase relaxation rate to improve imaging of oxygen;   introducing said paramagnetic contrast agent into a living sample to be imaged;   providing a magnetic resonator;   placing said living sample within the magnetic resonator;   generating a first series of RF excitation pulses, having an RF frequency between about 200 and 400 MHz separated by time intervals greater than about 4 microseconds;   coupling each RF excitation pulse in said first series to said resonator to induce EPR in said sample while isolating the gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response radiation is generated in response to each excitation pulse in said first series to generate a first series of corresponding EPR response signals based on the interaction of in vivo oxygen with said paramagnetic contrast agent in time intervals between said first series of RF excitation pulses;   digitizing and summing said first series of EPR response signals to obtain accurate values of EPR response signals; and   processing said accurate value of said EPR response signals to generate a first series of EPR parameter signals.   
     
     
       5. The method of claim 4 further comprising the steps of: generating a second series of RF excitation pulses separated by time intervals greater than about 4 microseconds;   phase-shifting said second series of RF excitation pulses by 180° to generate phase-shifted pulses;   coupling each-phase shifted RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each phase-shifted pulse in said second series to generate a second series of corresponding EPR response signals based on the interaction of in vivo oxygen with said paramagnetic contrast agent in time intervals between said RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first series of EPR response signals to subtract systematic noise and DC bias to obtain accurate values of said EPR response signals; and   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals.   
     
     
       6. The method of claim 4 further comprising the steps of: generating a first gradient magnetic field along a first axis prior to generating said first series of RF excitation pulses and maintaining said field until after said first series of EPR response signals have been generated to form a first projection of said sample; and   generating a second gradient magnetic field along a second axis;   generating a second series of RF excitation pulses, subsequent to generating the second gradient magnetic field, separated by time intervals greater than about 4 microseconds;   coupling each RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated to generate a corresponding second series of EPR response signals, based on the interaction of in vivo oxygen with said paramagnetic contrast agent, in time intervals between said RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first series of EPR response signals to subtract systematic noise and DC bias to obtain accurate values of said EPR response signals; and   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals and form a second projection of said sampler.   
     
     
       7. A method for measuring EPR parameters utilized to perform pulsed EPR measurement or imaging of a sample placed within a magnetic resonator which excites the sample when an RF radiation pulse is received to induce the sample to emit response RF radiation subsequent to excitation, with a gated RF amplifier for amplifying response RF radiation to form an EPR response signal, said method comprising the steps of: generating a corresponding first series of pseudo-random numbers having either a first or second value;   generating a first series of RF excitation pulses, having an RF frequency between about 200 and 400 MHz and separated time intervals greater than about 4 microseconds;   phase-shifting each pulse in said first sequence by 180° if a corresponding number in said first series has said second value and transmitting each pulse without phase-shift if a corresponding number in said first sequence is equal to said first value to generate a first series of phase-processed excitation pulses pulses;   coupling each phase-processed RF excitation pulse in said first series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each phase-processed pulse in said first series to generate a corresponding first set of EPR response signals based on paramagnetic resonance in said sample in time intervals between said first series of phase-processed RF excitation pulses;   digitizing and summing said first series of EPR response signals to obtain accurate values of said EPR response signals; and   performing a Hadamard transformation on obtained EPR response signals to obtain free induction decay parameters.   
     
     
       8. A method for measuring EPR parameters utilized to perform in vivo measurement of free radicals in a sample comprising the steps of: providing a living sample;   providing a magnetic resonator;   placing said living sample within the magnetic resonator;   generating a first series of RF excitation pulses, having an RF frequency between about 200 and 400 MHz and separated time intervals greater than about 4 microseconds;   coupling each RF excitation pulse in said first series to said resonator to induce EPR in said sample while isolating the gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response radiation is generated in response to each excitation pulse in said first series to generate a first set of corresponding EPR response signals, based on in vivo paramagnetic resonance of free radicals in said sample, in time intervals between said first series of RF excitation pulses;   digitizing and summing said first series of EPR response signals to obtain accurate values of said EPR response signals; and   processing said accurate values of EPR response signals to generate a first series of EPR parameter signals.   
     
     
       9. The method of claim 8 further comprising the steps of: generating a second series of RF excitation pulses separated intervals greater than about 4 microseconds;   phase-shifting said second series of RF excitation pulses by 180° to generate phase-shifted RF excitation pulses;   coupling each-phase shifted RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each phase-shifted pulse in said second series to generate a corresponding second series of EPR response signals, based on in vivo paramagnetic resonance of free radicals in said sample, in time intervals between said phase-shifted RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first series of EPR response signals to subtract systematic noise and DC bias to obtain accurate values of said EPR parameters; and   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals.   
     
     
       10. The method of claim 8 further comprising the steps of: generating a first gradient magnetic field along a first axis prior to generating said first series of RF excitation pulses and maintaining said field until after said first series of EPR response signals have been generated to form a first projection of said sample; and   generating a second gradient magnetic field along a second axis;   generating a second series of RF excitation pulses, subsequent to generating the second gradient magnetic field, separated by time intervals greater than about 4 microseconds;   coupling each phase shifted RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated to generate a corresponding second series of EPR response signals, based on in vivo paramagnetic resonance of free radicals in said sample, in time intervals between said RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first set of EPR response signals to subtract systematic noise and DC bias to obtain accurate values of said EPR response signals; and   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals and form a second projection of said sample.   
     
     
       11. The method of claim 8 further comprising the steps of: providing a spin trapping agent; and   introducing said spin trapping agent into said sample to stabilize said free radicals for imaging.   
     
     
       12. A method for measuring EPR parameters utilized to perform pulsed EPR measurement or imaging of a sample placed within a magnetic resonator which excites the sample when an RF radiation pulse is received to emit response RF radiation subsequent to excitation, with a gated RF amplifier for amplifying response RF radiation to form an EPR response signal, said method comprising the steps of: generating a corresponding first series of pseudo-random numbers having either a first or second value;   generating a first series of RF excitation pulses, having an RF frequency between about 200 and 400 MHz separated by time intervals greater than about 4 microseconds;   modulating the amplitude each RF excitation pulse in said first sequence to an OFF value if a corresponding number in said first series has said first value and to an ON value if a corresponding number in said first sequence has said second value to generate a first series of modulated RF excitation pulses;   coupling each RF excitation pulse in said first series of modulated RF excitation pulses to said resonator to induce EPR in said sample while isolating the gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to said first series of modulated RF excitation pulses to generate a corresponding first set of EPR response signals based on paramagnetic resonance in said sample in time intervals between said first series of modulated RF excitation pulses;   digitizing and summing said first series of EPR response signals to obtain accurate values of said EPR response signals; and   performing a Hadamard transformation on obtained EPR response signals to obtain free induction decay parameters.   
     
     
       13. A method for measuring EPR parameters utilized to perform RF FT EPR microscopy of free radicals or a paramagnetic contrast agent in a living sample placed within a magnetic resonator which excites the sample when an RF radiation pulse is received to emit response RF radiation subsequent to excitation, with a gated RF amplifier for amplifying response RF radiation to form an EPR response signal, said method comprising the steps of: generating a first series of RF excitation pulses, having an RF frequency between about 200 and 400 MHz separated by time intervals greater than about 4 microseconds;   coupling each RF excitation pulse in said first series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from the resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each excitation pulse in said first series to generate generating a first set of EPR response signals based on in vivo paramagnetic resonance of free radicals in said sample in time intervals between said first series of RF excitation pulses;   digitizing and summing said first series of EPR response signals to obtain accurate values of EPR response signals; and   processing said first series of EPR response signals to generate a first series of EPR parameter signals.   
     
     
       14. The method of claim 13 further comprising the steps of: generating a second series of RF excitation pulses separated by time intervals greater than about 4 microseconds;   phase-shifting said second series of RF excitation pulses by 180° to form phase-shifted RF excitation pulses;   coupling each phase-shifted RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating the gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each excitation pulse in said second series phase-shifted RF excitation pulses to generate a second series of EPR response signals based on paramagnetic resonance of in vivo free radicals in time intervals between said RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first series of EPR response signals to subtract noise and DC signals to obtain accurate values of said EPR response signals   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals.   
     
     
       15. The method of claim 13 further comprising the steps of: generating a first gradient magnetic field along a first axis prior to generating said first series of RF excitation pulses and maintaining said field until after said first series of EPR response signals have been generated to form a first projection of said sample; and   generating a second gradient magnetic field along a second axis;   generating a second series of RF excitation pulses, subsequent to generating the second gradient magnetic field, separated by time intervals greater than about 4 microseconds;   coupling each phase shifted RF excitation pulse in said second series to said resonator to induce EPR in said sample while isolating said gated RF amplifier from said resonator;   coupling said gated RF amplifier to said resonator when said response RF radiation is generated in response to each excitation pulse in said first second to generate a corresponding second series of EPR response signals based on in vivo paramagnetic resonance of free radicals in said sample in time intervals between said RF excitation pulses in said second series;   digitizing and subtracting said second series of EPR response signals from said first set of EPR response signals to subtract noise and DC signals to obtain accurate values of said EPR parameters; and   processing said accurate values of said EPR response signals to generate a second series of EPR parameter signals and form a second projection of said sample.   
     
     
       16. The method of claim 13 further comprising the steps of: providing a spin trapping agent;   introducing said spin trapping agent into said sample to stabilize said free radicals for imaging.   
     
     
       17. A fast response pulsed radiofrequency (RF) electron paramagnetic resonance (EPR) imaging system for forming an EPR image of a sample, said imaging system coupled to an RF signal generator that provides an RF excitation signal and an RF reference signal, with the RF signal having a frequency range of about 200 to 400 Mhz and receiving a system clock signal, said system comprising: a pulse generator, having an input coupled to receive the system clock signal and said RF excitation signal, for generating sequential, non-overlapping transmit, diplexer, receive, and Q-switching gating pulses;   a gating circuit, coupled to receive RF radiation and coupled to receive a transmit gating pulse having a duration of about 10 to 90 nanosecond, for transmitting said an RF signal when said transmit gating pulse is asserted, to form an RF excitation pulse having a duration of about 10 to about 90 nanoseconds with rise times of less than about 2 nanoseconds;   an ultra-fast data acquisition system including: a gated amplifier, having a signal input port and having a control input for receiving a receive gating pulse, said gated amplifier for amplifying RF radiation received at said signal input port only when said receive gating pulse is received and said gated amplifier being isolated from RF radiation received at said signal input port when said receive gating pulse is not received, with said gated amplifier for amplifying EPR response RF radiation received at said signal input port to form an EPR response signal;   demodulating means, coupled to receive said EPR response signal and said RF reference signals, for demodulating said EPR response signal to form an EPR parameter signal;   an ultra-fast, sampling and summing unit, for averaging a series of EPR parameter signals to increase signal to noise ratio, said sampling and summing unit including a high-speed sampler to digitize each received EPR parameter signal and summing means, coupled to receive each digitized EPR parameter signal, for generating a running sum of said digitized EPR parameter signals; and     a resonator for inducing paramagnetic resonance in a sample when an excitation pulse is received, for detecting EPR response RF radiation emitted from the sample due to paramagnetic resonance, and for outputting EPR response RF radiation;   a diplexer, coupled to said to receive said RF excitation pulse, coupled to said resonator to receive the EPR response RF radiation, coupled to the signal input port of said gated amplifier, and having a control input for receiving the diplexer gating pulse of a preset duration, said diplexer for coupling said RF excitation pulse to said resonator when said diplexer gating pulse is received, for isolating said RF excitation pulse from said ultra-fast data acquisition system when said diplexer gating pulse is not received, and for providing said EPR response RF radiation from said resonator to the input signal port of said gate amplifier subsequent to receiving said diplexer gating pulse; and   Q-switching means, coupling said resonator to said diplexer and coupled to said pulse generating circuit to receive said Q-switching gating pulse, for increasing resonator Q decreasing the ring-down time of the resonator.   
     
     
       18. The system of claim 17 further comprising: a phase shifter, coupled to receive said RF signal and having an output coupled to said gating circuit, for controllably either passing or phase-shifting said RF signal by 180°.